Air pollution is a pervasive environmental threat estimated to cause ~800,000 deaths every year worldwide, mostly due to cardiovascular disease. This proposal addresses a fundamental mechanistic and pharmacologic question about effects of air pollution, which can most effectively be addressed through controlled human exposure experiments: does exposure have epigenetic effects that may have downstream subclinical or clinical consequences, and can adverse effects be safely reduced pharmacologically? Consistent evidence from in- vitro and human studies have shown that exposure to air particulate matter pollution (PM, i.e., fine particles) induces hypomethylation of the DNA, an epigenetic process that can underlie the activation of inflammatory genes and is postulated to link inhalation of PM into the lungs with cardiovascular inflammation and adverse responses. Our goal is to determine whether a pharmacological intervention with methyl-donors (i.e., folic acid, Vitamins B6 & B12, betaine, methionine, and choline) can avert this DNA methylation loss and mitigate the cardiovascular effects induced by PM exposure. We will use experiments of human controlled exposure to PM - which reproduce conditions of exposure similar to those found in real life in urban environments - to conduct a randomized, double-blind, placebo-controlled crossover study. We will test whether pharmacological intervention with methyl-donors attenuates the effects of PM exposure on DNA methylation (Aim 1), mRNA expression & plasma cytokines (Aim 2), and blood pressure, arterial vasoconstriction, endothelial function, and autonomic control of the heart (Aim 3). Our study is poised to be the first human investigation to translate a wealth of animal data showing that methyl-donors can be used to modulate epigenetic states and avert environmental effects. We have a unique opportunity to achieve this goal because we have access to one of the few facilities worldwide for human controlled-exposure studies, as well as to state-of-the-art resources for epigenetics investigations. We will examine DNA methylation and mRNA expression in T-helper cells from human individuals, a cell type with key roles in determining adverse hypertensive and endothelial responses, as shown in several animal models. We will test the effects of methyl-donors on a battery of cardiovascular endpoints that are highly sensitive to PM exposure. We will explore the use of advanced statistical methods for mediation analyses to understand the relationships among PM, DNA methylation, RNA expression, plasma cytokines, and cardiovascular endpoints. The study will be conducted by an investigative team that has conducted seminal work in all of the research areas on which this proposal is built upon, including environmental epigenetics, cardiovascular effects of PM, and human controlled exposure studies.